Independently of the manual method for counting and analysing a leucocytic sub-population which consists in visualising by means of a microscope blood samples spread on a slide, and previously coloured, there exist automatic methods which perform much better and are more accurate for simultaneously differentiating several types of blood populations. Two types of analysis methods are essentially known : the methods of resistivity size analysis and optical analysis methods.
Analysis and automatic counting by resistivity are based on the principle of a cell passing through an electric field where a constant current is maintained. The resistance which this cell opposes in the field causes an increase of the voltage required for the constancy of the current in accordance with Ohm's law. The voltage pulse generated by the passage of this cell is proportional to the resistance opposed, so to its volume without consideration of form. In order to determine a leucocytic sub-population using this principle, it is necessary to treat the blood sample previously with specific cytochemical agents for partially destroying the cells not under consideration, in order to obtain a size discrimination as accurate as the treatment is specific.
The differentiation method the most commonly used with this principle is a method called "leucocytic screening", for obtaining an approximation of the blood formula over three populations : the Lymphocytes, the Mononucleates and the Granulocytes.
The essential part of the analysis is based on the use of a lytic reagent with differential action. Because of this reagent, the membranes of the leucocytes let the contents of the cytoplasm escape. Since the cells have no granules contained in their cytoplasm, they have then the cytoplasmic membrane covering their nucleus; the granulocytes keep a part of the cytosol, their granules partially preventing its escape.
The main drawback of this method resides in the fact that the leucocytes are only differentiated by their final size and that the action of the lysis on certain cells, particularly the eosinophils and the basophils is not completely under control, which frequently causes overlapping, even superimposition of the populations which makes differentiation impossible or very hazardous.
Furthermore, the analysis and counting of a leucocytic sub-population using optical methods is based either on the principle of optical diffraction measurement, or on the principle of measurement of the optical density of a cell, or a combination of the two.
In the first case, a cell passing through a light ray generates diffraction of the incider light, whose intensity at different angles depends on one size of the cell and on the amount of light absorbed thereby. An optical collector having at its center a disc with a black background is placed in the alignment of the optical path. The light transmitted is stopped by the disc whereas the diffracted light is collected on a photosensitive sensor whose response is proportional to the diffraction of the cell.
In the second case, a cell passing through a light ray generates absorbance of the incident light. The transmitted light is filtered at the wavelength corresponding to the coloration of the cell, then is collected by a photosensitive sensor whose response is proportional to the light absorbed at the specified wavelength.
Since the cellular diffraction is related to the absorbance of the cell as well as to its form, measurements of volume remain uncertain, particularly in so far as the leucocytes are concerned, and it is necessary to make the cells to be measured artificially spherical. It can also be noted that a very coloured large sized cell will be seen smaller than its actual size.
Combinations of these principles are used so as to obtain, for the same cell, values of diffraction and coloration intensity or diffraction values at different wavelengths, or else diffraction values at different angles. By means of specific cytochemistries, cellular discrimination can be obtained.
The apparatus using these principles suffer from great sensitivity of the optical alignment which means that the diffraction measurements have only relative stability. Numerous factors also cause the measurement to be fragile such, in particular, as fouling up of the reading tank, the sensitivity of the optical parts to atmospheric dust and to the temperature and hygrometry of the premises. Moreover, the high technicity required for a well performing optical assembly makes the cost of the apparatus unattractive.